Ca²⁺ oscillations can be induced in mammalian eggs and somatic cells by microinjection of a cytosolic sperm protein factor. The nature of the sperm factor-induced Ca²⁺ signaling was investigated by adding sperm protein extracts to homogenates of sea urchin eggs, which contain multiple classes of Ca²⁺ release mechanisms. We show that the sperm factor mobilizes Ca²⁺ from non-mitochondrial Ca²⁺ stores in egg homogenates after a distinct latency. This latency is abolished by preincubation of sperm extracts with egg cytosol. The preincubation step is highly temperature-dependent and generates a high molecular weight, protein-based Ca²⁺-releasing agent that can also mobilize Ca2+ from purified egg microsomes. This Ca²⁺ release appears to be mediated via both inositol 1,4,5-trisphosphate and ryanodine receptors, since homologous desensitization of these two release mechanisms by their respective agonists inhibits further release by the sperm factor. However, sperm factor-induced Ca²⁺ release by these channels is independent of inositol 1,4,5-trisphosphate or cADPR since antagonists of either of these two messengers did not block the Ca²⁺ release effected by the sperm factor. The sperm protein factor may cause Ca²⁺ release via an enzymatic step that generates a protein-based Ca²⁺-releasing agent.